饲料高铜、锌和抗生素对断奶仔猪肠道微生物耐药应答的协同作用研究

In order to prevent diarrhea and to promote growth of weaning piglets, antibiotics, copper and zinc were supplemented into feed. Copper and zinc is essential trace element required for vital biological functions in both prokaryotic and eukaryotic cells. However, copper and zinc in excess concentrations are toxic to cells because they induce the production of intracellular reactive oxygen radicals, which inactivate cell components such as nucleic acids, lipids, and proteins. Therefore, cells tightly regulate intracellular copper and zinc concentrations to avoid toxicity. To survive, many bacterial species have developed copper resistance. Acquired copper resistance mediated by the plasmid-localized tcrB gene has been described for E. faecium and E. faecalis isolated from both production animal and humans in several European countries. A close genetic link between tcrB and glycopeptide-resistance (vanA) has been demonstrated for E. faecium. The objective of this study is to describe the relationship between metal-resistance and antibiotic-resistance for gut microbiota of weaned piglets, to answer whether in-feed copper and zinc at high concentrations could drive co-selection of antibiotic resistance in piglets' gut. 48 of weaned piglets will be divided into four groups, control group 1 (copper and zinc at low concentrations without antibiotic), control group 2 (copper and zinc at low concentrations with antibiotic), treatment group 1 (copper and zinc at high concentrations without antibiotic), and treatment group 2 (copper and zinc at high concentrations with antibiotic). Fecal samples will be used to analyze the effect of in-feed antibiotic, copper and zinc on composition, metal-resistance gene and antibiotic-resistance gene levels of gut microbiota. Isolated and identified E. coil and E. faecium will be used to determine antibiotic susceptibility, copper and zinc susceptibility, tcrB gene and parC gene. The tcrB-positive/ negative, and tcrB-positive/ negative E. coil /E. faecium will be incubated in vitro to demonstrate the co-selection of antibiotic and metal resistance.

为防断奶仔猪腹泻和促生长，饲料中添加抗生素和高铜、锌是常用的营养策略。铜、锌是宿主细胞和肠道细菌正常生长所必须，但是高水平的铜、锌却能杀菌并诱导细菌产生抗性应答。肠道细菌重金属抗性与抗生素耐药性的关系如何？饲料高铜、锌是否加剧了断奶仔猪肠道细菌的耐药问题，寻求控制肠道耐药菌产生的有效营养措施是本研究的目的和意义所在。断奶仔猪分为四组，分别饲喂无抗低铜、锌日粮、有抗（恩诺沙星）低铜、锌日粮、无抗高铜、锌日粮和有抗高铜、锌日粮，采集粪便分析恩诺沙星和高铜、锌引起的菌群结构和耐药、重金属抗性基因变化；分离并测定大肠杆菌和粪肠球菌的抗生素和铜、锌敏感性，以阐明饲料抗生素和高铜、锌在肠道细菌抗生素耐药性形成中的协同作用。体外培养不同敏感型大肠杆菌和粪肠球菌，分析恩诺沙星和高铜、锌引起的外排泵基因、外膜蛋白基因、parC基因、tcrB基因等表达量的变化，探讨高铜、锌和抗生素协同诱导耐药应答的可能机制。

2006年欧盟全面禁用抗生素促生长剂后，饲料添加高铜、锌等微量元素随之成为普遍采用的替代措施，200-250 mg·kg-1铜对猪生产性能的影响与抗生素相似，虽机制不明日粮高铜可能共选择抗生素耐药菌。我国在动物生产中限制或停止抗生素促生长使用和规范抗生素治疗使用的步伐正在加快，2015年底禁用了氧氟沙星等4种喹诺酮类兽药，2016年底禁止硫酸黏杆菌素用于饲料添加剂，尽管如此，我国当前养猪生产中仍然普遍同时使用抗生素和高铜，所面对的抗生素耐药问题更为复杂和严峻。本课题首先以哺乳仔猪为对象，以大肠杆菌、肠球菌和乳酸杆菌为肠道细菌代表，比较了饲料中抗生素和高铜存在与否对其肠道细菌耐药水平的影响，结果表明菌株铜抗性水平与恩诺沙星耐药率显著正相关，而撤除哺乳仔猪教槽料中抗生素和无机铜可显著降低头孢噻肟耐受大肠杆菌数和比例，并显著提高大肠杆菌的铜敏感性。进一步以长期重复使用的发酵床垫料为对象，验证了饲料源抗生素和重金属对细菌耐药性进化的协同影响。在此基础上，以耐药背景相对较低的巴马香猪为对象，给与饲料高铜和亚治疗剂量恩诺沙星处理，发现其肠道大肠杆菌、肠球菌和乳酸杆菌的恩诺沙星耐受和铜抗性间存在明显的叠加效应，高铜组分离菌株对恩诺沙星的耐受性高于低铜组分离菌株，菌株对恩诺沙星及铜的耐受性之间存在一定的联系。同时以大鼠和哺乳仔猪为对象，给与饲料高铜和治疗剂量头孢噻肟处理，平板计数和S1-PFGE及Southern杂交等分子分析表明，高铜促进了体外和体内blaCTX-M-1阳性质粒的水平传播和维持，对加速耐药基因的传播具有一定的促进作用。为综合考虑铜的吸收和对动物健康及细菌耐药性传播的潜在风险，本课题系统研究了不同铜水平（6、20、120、240和300 mg·kg-1）对大鼠和仔猪生长健康的影响，发现高铜（120、240和300 mg·kg-1）对大鼠和仔猪生长健康不利；6mg·kg-1铜是AIN93G和NRC（2012）推荐的仔猪铜需要量，但在我国当前的生产条件下不能完全满足动物生长需要；20 mg·kg-1日粮铜水平可以满足动物生长和生理活动需求，建议在我国当前的教槽料和前期保育料中使用20 mg·kg-1的日粮铜。